Nicotinamide Mononucleotide Adenylyltransferase 1 Regulates Cerebral Ischemia-Induced Blood–Brain Barrier Disruption Through NAD+/SIRT1 Signaling Pathway

The molecular mechanisms of blood–brain barrier (BBB) disruption in the early stage after ischemic stroke are poorly understood. In the present study, we investigated the potential role of nicotinamide mononucleotide adenylyltransferase 1 (NMNAT1) in ischemia-induced BBB damage using an animal middle cerebral artery occlusion (MCAO) model of ischemic stroke. Recombinant human NMNAT1 (rh-NMNAT1) was administered intranasally and Sirtuin 1 (SIRT1) siRNA was administered by intracerebroventricular injection. Our results indicated that rh-NMNAT1 reduced infarct volume, improved functional outcome and decreased BBB permeability in mice after ischemic stroke. Furthermore, rh-NMNAT1 prevented the loss of tight junction proteins (occludin and claudin-5) and reduced cell apoptosis in ischemic microvessels. NMNAT1-mediated BBB permeability was correlated with the elevation of nicotinamide adenine dinucleotide (NAD+)/NADH and SIRT1 level in ischemic microvessels. In addition, rh-NMNAT1 treatment significantly decreased the levels of acetylated nuclear factor-κB, acetylated p53 and matrix metalloproteinase-9 in ischemic microvessels. Moreover, the protective effects of rh-NMNAT1 were reversed by SIRT1 siRNA. In conclusion, these findings indicate that NMNAT1 protects BBB after ischemic stroke in mice which was in part via the NAD+/SIRT1 signaling pathway in brain microvascular endothelial cells. NMNAT1 may be a novel potential therapeutic target for reducing BBB disruption after ischemic stroke.

Nicotinamide mononucleotide adenylyltransferase uses its NAD+ substrate-binding site to chaperone phosphorylated Tau

Tau hyper-phosphorylation and deposition into neurofibrillary tangles have been found in brains of patients with Alzheimer’s disease (AD) and other tauopathies. Molecular chaperones are involved in regulating the pathological aggregation of phosphorylated Tau (pTau) and modulating disease progression. Here, we report that nicotinamide mononucleotide adenylyltransferase (NMNAT), a well-known NAD+ synthase, serves as a chaperone of pTau to prevent its amyloid aggregation in vitro as well as mitigate its pathology in a fly tauopathy model. By combining NMR spectroscopy, crystallography, single-molecule and computational approaches, we revealed that NMNAT adopts its enzymatic pocket to specifically bind the phosphorylated sites of pTau, which can be competitively disrupted by the enzymatic substrates of NMNAT. Moreover, we found that NMNAT serves as a co-chaperone of Hsp90 for the specific recognition of pTau over Tau. Our work uncovers a dedicated chaperone of pTau and suggests NMNAT as a key node between NAD+ metabolism and Tau homeostasis in aging and neurodegeneration.

Development of a Bioluminescent High-Throughput Screening Assay for Nicotinamide Mononucleotide Adenylyltransferase (NMNAT)

Nicotinamide mononucleotide adenylyltransferase (NMNAT; EC 2.7.7.1) catalyzes the reversible production of NAD+ from NMN+ and ATP and is a potential drug target for cancer and neurodegenerative diseases. A sensitive bioluminescent assay format suitable to high-throughput screening (HTS) and mechanistic follow-up has not been reported and is of value to identify new modulators of NMNATs. To this end, we report the development of a bioluminescent assay using Photinus pyralis ATP-dependent luciferase and luciferin for NMNAT1 in a 384-well plate format. We also report a mechanistic follow-up paradigm using this format to determine time dependence and competition with substrates. The assay and follow-up paradigm were used to screen 912 compounds from the National Cancer Institute (NCI) Mechanistic Diversity Set II and the Approved Oncology Set VI against NMNAT1. Twenty inhibitors with greater than 35% inhibition at 20 µM were identified. The follow-up studies showed that seven actives were time-dependent inhibitors of NMNAT1. 2,3-Dibromo-1,4-naphthoquinone was the most potent, time-dependent inhibitor with IC50 values of 0.76 and 0.26 µM for inhibition of the forward and reverse reactions of the enzyme, respectively, and was shown to be NMN and ATP competitive. The bioluminescent NMNAT assay and mechanistic-follow-up will be of use to identify new modulators of NAD biosynthesis.